Exploring relationships among multi-disciplinary assessments for knee joint health in service members with traumatic unilateral lower limb loss: a two-year longitudinal investigation

Motivated by the complex and multifactorial etiologies of osteoarthritis, here we use a comprehensive approach evaluating knee joint health after unilateral lower limb loss. Thirty-eight male Service members with traumatic, unilateral lower limb loss (mean age = 38 yr) participated in a prospective, two-year longitudinal study comprehensively evaluating contralateral knee joint health (i.e., clinical imaging, gait biomechanics, physiological biomarkers, and patient-reported outcomes); seventeen subsequently returned for a two-year follow-up visit. For this subset with baseline and follow-up data, outcomes were compared between timepoints, and associations evaluated between values at baseline with two-year changes in tri-compartmental joint space. Upon follow-up, knee joint health worsened, particularly among seven Service members who presented at baseline with no joint degeneration (KL = 0) but returned with evidence of degeneration (KL ≥ 1). Joint space narrowing was associated with greater patellar tilt (r[12] = 0.71, p = 0.01), external knee adduction moment (r[13] = 0.64, p = 0.02), knee adduction moment impulse (r[13] = 0.61, p = 0.03), and CTX-1 concentration (r[11] = 0.83, p = 0.001), as well as lesser KOOSSport and VR-36General Health (r[16] = − 0.69, p = 0.01 and r[16] = − 0.69, p = 0.01, respectively). This longitudinal, multi-disciplinary investigation highlights the importance of a comprehensive approach to evaluate the fast-progressing onset of knee osteoarthritis, particularly among relatively young Service members with lower limb loss.


Participants
Thirty-eight male SM with unilateral lower limb loss (23 transfemoral and 15 transtibial; mean ± standard deviation age: 37.9 ± 6.5 yr; stature: 1.78 ± 0.05 m; body mass: 89.2 ± 18.4 kg; time since injury: 114.4 ± 70.9 months) were enrolled into a prospective, two-year longitudinal study utilizing a comprehensive evaluation (i.e., clinical imaging, gait biomechanics, physiological biomarkers, and patient-reported outcomes) for identifying the onset, progression, and overall impact of knee OA on functionality and quality of life; eighteen (9 transtibial and 9 transfemoral) SM returned for the two-year follow-up.One participant was removed from subsequent analyses due to receiving a medial knee arthroplasty between baseline and follow-up; thus, seventeen SM with unilateral lower limb loss (9 transtibial and 8 transfemoral) were included (Fig. 1).The Walter Reed National Military Medical Center Institutional Review Board approved all experimental procedures (protocol #500081), performed in accordance with all relevant guidelines and regulations, and written informed consent was obtained from all participants prior to testing.

Gait biomechanics
All SM at baseline (n = 17) and two-year follow-up (n = 17) completed a gait evaluation, walking at a self-selected pace along a 15 m walkway.Full-body kinematics were obtained using an 18-camera motion capture system (Qualisys, Göteborg, SE) to track (120 Hz) the positions of 62 reflective markers.Markers were placed on the head (× 4), C7 and T10 spinal processes, sternal notch, xiphoid, bilaterally across the acromion, humeri, lateral elbows, forearm, radii, ulnae, dorsal hand, posterior and anterior superior iliac spines, calcanei, hallux, and the 2nd and 5th metatarsals.Cluster-based tracking markers were also placed bilaterally on each thigh and shank (4 markers per cluster).Twelve additional calibration-only markers were placed bilaterally on the medial epicondyle of the elbow, lateral and medial epicondyles of the knee and ankle maleoli, and the medial aspect of the 1st metatarsal.Bilateral ground reaction forces were simultaneously sampled (1200 Hz) from six force platforms (AMTI, Watertown, MA, USA) embedded within the walkway.Marker trajectories and ground reaction forces were low-pass filtered (6 and 45 Hz, respectively) with a dual-pass, 2nd order Butterworth filter.
Temporal-spatial parameters, knee joint kinematics, and knee joint kinetics were calculated in Visual3D (C-Motion, Germantown, MD, USA).Briefly, bilateral gait events (i.e., heel strike and toe off) were first determined using a foot kinematic-based detection algorithm 23 ; temporal-spatial outcomes were derived (walking speed, stride length, stride width, and cadence).Peak sagittal knee angles from each gait cycle were extracted to compute joint range of motion (ROM).Joint angles were computed using a flexion-extension, ab/adduction, axial rotation sequence.Inverse dynamics were used to calculate external knee adduction moment (KAM) and knee flexion moment (KFM), resolved relative to the proximal (thigh) segment; peak KAM and KFM were extracted, as were KAM impulse (time integral of KAM) and KAM loading rate (slope between 20 and 80% of time from heel strike to first peak).Knee joint contact forces (total, medial, and lateral) were calculated based on the model of Schipplein and Andriacchi, and developed as described in previous literature 24,25 .All joint kinematic and kinetic parameters are only reported for the contralateral limb (i.e., non-limb loss).

Physiological biomarkers
Fifteen SM provided serum samples at baseline and follow-up, while four (of eight at baseline) SM opted to have synovial fluid samples taken at follow-up.Whole blood samples were collected through venipuncture; serum www.nature.com/scientificreports/ was obtained through centrifugation at 1000RPM for 10 min at room temperature.Synovial fluid was aspirated from the contralateral knee by a trained physician.After collection, both biofluids were aliquoted and stored at − 80 °C for downstream analyses.Subsequently, serum and synovial fluid samples were analyzed for collagen II cleavage (C2C), N-Propeptide of Collagen IIA (PIIANP), hyaluronic acid (HA), and cartilage oligomeric matrix protein (COMP) as markers of osteochondral (bone and cartilage) remodeling; and C-Telopeptide of Type 1 Collagen (CTX-1) and N-telopeptide of Type 1 Collagen (NTX-1) as markers of subchondral bone degradation via Enzyme-Linked Immunosorbent Assays according to manufacturer's protocol.Serum and synovial fluid samples were also analyzed for various inflammatory and tissue-remodeling markers via multiplex bead assays.For markers related to collagen and mineral metabolism, custom multiplex kits (Invitrogen) were used to evaluate the following markers: matrix metalloprotease (MMP) -2, MMP3, MMP7, MMP8, MMP9, MMP12, MMP13, and tissue-inhibitor or metalloprotease (TIMP)-1.Specifically, for cytokines and chemokines, a Procar-taPlex Human Cytokine/Chemokine kit (Invitrogen, Carlsbad, CA, USA) was used according to manufacturer recommendations.

Statistical analyses
Statistical analyses were performed using Statistical Package for Social Science (SPSS) software (version 25; Chicago, IL, USA).Sample characteristics are presented as numbers, percentages, means ± standard deviations.Normal distribution was evaluated using the Shapiro-Wilk tests.To first compare outcomes between timepoints (i.e., baseline vs. two-year follow-up), paired sample t-tests, or Wilcoxon signed-rank tests (where applicable), were used to compare all outcomes within each domain: (i) demographics, (ii) radioanatomy, (iii) gait biomechanics, (iv) physiological biomarkers, and (v) patient-reported.Effect sizes are reported using Cohen's d.Chisquare analysis was utilized to compare the proportions of KL classifications.Second, associations between each outcome (at baseline) with changes in tibiofemoral and patellofemoral joint space (from baseline to two-year follow-up) were investigated using Pearson's correlations.Kendall's tau-b correlation was utilized for correlations between Insall-Salvati proportions and joint space narrowing.Values of p < 0.05 were considered significant.

Discussion
This comprehensive, longitudinal evaluation for knee joint health in SM with unilateral lower limb loss aimed to identify relationships among associative risk factors at baseline with knee joint space narrowing.The primary hypotheses that decreases in medial tibiofemoral and patellofemoral joint spaces will be associated with increased knee joint kinetics and elevated serum concentrations, and synovial fluid inflammatory biomarkers of bone and cartilage metabolites, were partially supported.Radiographically, KL grading suggests overall minimal to moderate joint degeneration (i.e., KL ≤ 3), with worsening of knee joint health from baseline to two-year follow-up (17-42% with KL ≥ 2).Of the seven SM (2 TT, 5 TF) who presented at baseline with no clinical joint degeneration (KL = 0), all returned at two-year follow-up with evidence of degeneration (KL ≥ 1) within the contralateral knee.Although ultimately excluded at follow-up, one participant (42 yrs of age) received a medial knee arthroplasty during the study period (KL = 3 at baseline, with evidence of degeneration in the medial [Outerbridge Grade = 4], lateral [Outerbridge Grade = 2], patellofemoral [Outerbridge Grade = 3] compartments).A secondary hypothesis predicted that radioanatomic measures (e.g., bisect offset, patellar tilt, and tibiofemoral joint angle) would be associated with more significant medial patellofemoral joint space narrowing; this hypothesis was partially supported, as patellar tilt was positively associated with medial and lateral patellofemoral joint space narrowing, consistent with prior literature in persons www.nature.com/scientificreports/with patellofemoral pain and OA progression 18,29 .Furthermore, decreases in the Insall-Salvati ratio, a patellar height measurement, were negatively associated with medial and lateral tibiofemoral joint space narrowing.While the ratio of participants with patella alta/baja was similar between time points, changes in the Insall-Salvati ratio can impact patellofemoral joint mechanisms and often result in diminished function and deterioration of knee joint health 30 .Changes in patellar tendon length has been extensively studied following anterior cruciate ligament repairs and total knee arthroplasty, but not in the population of SM with limb loss, who present a  unique morphological change, absent surgical intervention 31 .Our results concur with the literature, noting that longer patellar tendons (larger Insall-Salvati ratio) present a situation wherein the patella may be unstable and maltrack, impacting the articular surfaces of the knee and patella 32 .Within our study sample, decreases in the patellar height could suggest a more proximal patellar positioning, prior to study enrollment.Some potential explanations for a decreased Insall-Salvati ratio may be due to increases in weight or forces upon the contralateral knee, or decreases in the retropatellar fat pad [33][34][35] .Although literature notes a higher prevalence of patella baja with greater body mass 33 , it can be surmised that the increased dependence on the contralateral limb after unilateral limb loss may mimic similar overloading conditions of overweight/obesity.Further research is needed to longitudinally track morphological changes in knee radioanatomy and patellar anatomy of the contralateral knee immediately following limb loss, as our study population at enrollment were several years past their injury.Biomechanically, while cross-sectional evaluations indicate larger knee joint loads in persons with vs. without lower limb loss, here there was a general lack of changes between timepoints in the current study.Common biomechanical adaptations during walking with vs. without knee OA, though variable across studies 36 , include lesser knee flexion-extension excursion (largely due to joint stiffness or pain) or greater loading metrics (e.g., external KAM peak or loading rate).While prior studies have identified differences in joint loads with vs. without knee OA, such changes are more apparent with more severe stages of OA (i.e., KL ≥ 3) 37 , and peak KAM is positively correlated with the severity of knee OA (medial tibiofemoral joint space narrowing) 38,39 .Here, despite the similar (peak) joint loads between timepoints, and minimal-moderate joint degeneration (i.e., KL1-3), there were several moderate associations between KAM loading measures and tibiofemoral or patellofemoral joint space narrowing.We and others have suggested that idiopathic knee OA after limb loss results not necessarily from chronically high joint loads but rather from "unusual" joint loads, defined as loads the joint is not conditioned to sustain frequently 24,40,41 .Exposure to such unusual loads could be particularly hazardous to long-term joint health if introduced following an extended period of low activity and ostensible structural weakening, such as if an individual with unilateral limb loss hops on one leg (e.g., 42 ).However, more ecologically valid methods are needed to comprehensively track biomechanical and mobility outcomes for prolonged durations and across numerous activities, at home and in the community (vs.exclusively in a lab during controlled conditions).www.nature.com/scientificreports/Physiologically, while numerous serum biomarkers changed from baseline to follow-up, CTX-1 specifically was positively correlated with lateral patellofemoral joint space narrowing and highlights the presence of increased osteochondral breakdown in systemic biomarkers (i.e., serum).However, we must acknowledge that SM with limb loss often present with multi-site musculoskeletal pain 43,44 which may confound joint-specific musculoskeletal research, particularly in the interpretation of some systemic biomarkers and other outcomes (i.e., gait biomechanics, patient-reported function or quality of life).In the current sample, while participants reported multi-site pain (e.g., residual limb, low back), these did not change from baseline to two-year follow-up.Nevertheless, more specific to the knee joint, we identified increases in MMP-13 and MMP-8 among synovial fluid at follow-up, which were respectively associated with greater narrowing of the medial tibiofemoral and lateral patellofemoral joint spaces.These MMP biomarkers indicate joint catabolic activity, ultimately leading to the loss of cartilage via inhibition of cartilage differentiation and promotion of chondrocyte apoptosis 45,46 .This is a significant finding and novel contribution to both the limb loss and non-limb loss OA populations.
Patient-reported outcomes provide important insights on symptom severity and corresponding influences on well-being and quality of life; however, the ability to accurately assess pain severity is likely hampered in the SM population.For example, while pain experience can be subjective and thus difficult to compare between participants or populations, SM tend to under-report symptoms in both clinical and research settings 47 .Outcome measures such as the KOOS or VR-36 may be more appropriate to understand the impact and monitor changes in knee joint health among SM with limb loss.Here, KOOS scores generally indicated better outcomes (i.e., scores closer to 100) than reported in the literature across a range of OA pathologies 48 , for most subdomains.Also, only Symptoms and Quality of Life decreased from baseline to two-year follow-up, again noting a relatively lesser severity of degeneration within the current sample.VR-36 and SF-8A outcomes of the current sample provide a slightly different perspective from acute pain alone, with decreasing General Health from baseline to two-year follow-up, but similarly suggest overall better general health and lesser pain interference in comparison to existing literature 49,50 .

Limitations
Several limitations are present and should be considered.Acknowledging a general paucity of longitudinal data, many patients with knee OA enrolled in longitudinal studies do not progress radiographically during ~ two-year trials, highlighting that OA progression is highly variable by individual and stage of disease 9,51,52 ; in the absence of existing characterization of disease trajectory among SM with limb loss, we ultimately chose a two-year followup window to balance additional pragmatic considerations (i.e., attrition).Moreover, our broad cross-sectional recruitment strategy (i.e., no a priori grouping by joint health or other factors upon enrollment) generated a convenience sample with somewhat heterogeneous characteristics.Despite the likelihood for knee joint health to be influenced by time since injury and/or "severity" of injury (i.e., transtibial vs. transfemoral limb loss), neither the study design nor final dataset support the evaluation of such factors within the longitudinal framework.The prevalence of new amputations within the military has decreased every year since peaking in 2011 53 ; while our sample is more reflective of SM injured within this time period (time since injury = 119.8± 89.3 months), without baseline measures obtained more proximal (and/or prior) to time of injury we are unable to determine the causality of observed associations.While attrition is an inherent challenge for longitudinal studies, this challenge was magnified during the COVID-19 global pandemic as travel and research restrictions prevented many participants from returning for follow-up.Importantly, compared to participants who completed follow-up, participants lost to follow-up were not different by baseline demographics, medical history, or knee joint health grading (Supplementary Table 1); thus, mitigating potential confounding effects of attrition beyond sample size alone.

Conclusion
This study establishes a comprehensive approach for evaluating (contralateral) knee joint health after unilateral lower limb loss and emphasizes the need for early identification/intervention to mitigate the fast-progressing degeneration among SM with lower limb loss.In particular, SM with traumatic limb loss are typically younger at time of injury and more active following limb loss, increasing the likelihood of suffering long-term consequences of poor knee joint health on function and quality of life.Future research should continue to employ this multi-faceted approach to evaluate knee joint health and encourage the development of optimal interventions to maximize quality of life and long-term functional outcomes for SM with limb loss.

Table 1 .
Mean ± standard deviation participant demographics, joint space measurement, and knee joint health at baseline and 2-year follow-up.a Baseline and follow-up measures (n = 13).b Baseline and follow-up measures (n = 12).c Baseline and follow-up measures (n = 8).Bolded values with asterisks indicate significance (p ≤ 0.05).

Table 2 .
Mean ± standard deviation radioanatomic outcomes at baseline and 2-year follow-up, as well as correlations with changes (Δ) in tibiofemoral and patellofemoral joint space.Bolded values with asterisks indicate significance (p ≤ 0.05).

Table 3 .
Mean ± standard deviation gait biomechanical outcomes at baseline and 2-year follow-up, as well as correlations with changes (Δ) in tibiofemoral and patellofemoral joint space.Correlations in bold indicate p < 0.05.Correlation in bold and italicized indicate p < 0.01.Bolded values with asterisks indicate significance (p ≤ 0.05).

Table 4 .
Mean ± standard deviation blood serum biomarkers at baseline and 2-year follow-up, as well as correlations with changes in tibiofemoral and patellofemoral joint space.Bolded values with asterisks indicate significance (p ≤ 0.05).

Table 5 .
Mean ± standard deviation knee joint synovial fluid biomarkers at baseline and 2-year follow-up, as well as correlations with changes (Δ) in tibiofemoral and patellofemoral joint space.Bolded values with asterisks indicate significance (p ≤ 0.05).

Table 6 .
Mean ± standard deviation patient-reported outcomes at baseline and 2-year follow-up, as well as correlations with changes (Δ) in tibiofemoral and patellofemoral joint space.Bolded values with asterisks indicate significance (p ≤ 0.05).